The diverse biologic actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are thought to be mediated byintracellular receptor proteins which appear to operate at the level of the cell nucleus. We propose to continue our study of these unique polypeptides derived from both avian and mammalian sources utilizing immunologic as well as conventional probes. Investigation will focus upon four areas essential to our understanding of the molecular mechanism of 1,25(OH)2D3 action: 1) physical and biochemical properties of receptors, 2) intracellular biology of receptors and the role of 1,25(OH)2D3 3) functional characterization of receptors, and 4) molecular defects in receptors which confer cellular resistance to 1,25(OH)2D3. Physical and biochemical characterization will include evaluation of the two antigenically related forms of the avian receptor, topological mapping of both avian and mammalian receptors, determination of mammalian 1,25(OH)2D3-dependent receptor phosphoamino acid species, and isolation and sequence analysis of both avian and mammalian receptors. Aspects of the biology of 1,25(OH)2D3 receptors will include further evaluation of the intracellular origins of unoccupied forms, receptor synthesis and degradation, the effects of 1,25(OH)2D3 on cellualr levels of receptor and its mRNA, the nature and mechanism of receptor inactivation. Functional studies will include determination of potential receptor enzymatic activities, evaluation of teh functional significance of receptor modification, and analysis of receptor DNA sequence preference utilizing cloned vitamin D-dependent calbindin gene and receptor gene fragments. Evaluation of molecular abnormalities will involve the application of the above technology to the examination of abberrant human receptors from fibroblasts obtained from patients with reisitance to 1,25(OH)2D3 as well as from developed nonhuman variants. Collectively, the studies proposed here on normal receptors represent an integrated approach to the elucidation of the role of receptor in mediating various bilogical phenomena regulated by 1,25(OH)2D3. In addtion, they provide the basis for interpreting molecular defects in receptors which may be associated with vitamin D3-resistant disease states.
